The program proposed focusses on the development and application of improved techniques for synthesis and characterization of drug metabolites. We propose to exploit the use of immobilized microsomal enzymes for synthesis and mass spectrometry for characterization. We propose to continue to develop the chemistry of glucuronides, using our immobilized UDP-glucuronyl transferase system to synthesize C-linked, N-linked, N-hydroxyl-linked and stable isotope labelled glucuronides for chemical and physiochemical characterization. We propose to continue to study the stereoselectivity of our cell free UDP-glucuronyl transferase(s), and to examine the origins and structures of isomeric glucuronide conjugates which are formed with carboxylic acid groups. We propose to examine more fully the catalytic properties of our recently immobilized P-450 monooxygenase system, with respect to scope of effective substrates, positional selectivity, and stereochemical selectivity. We will compare the properties of our system to those known for microsomal and hepatic transformations, and also extend our system to new studies. Because both the monooxygenase and transferase systems are immobilized on the same beads and both activities can be supported simultaneously, we propose to evaluate glucuronide formation as a means to trap unstable oxygenated metabolites. Methodologic development will continue for both the synthetic and the analytical techniques.